间接酶联免疫吸附试验检测葡萄扇叶病毒的研究和应用

APPLICATION OF INDIRECT ELISA TO THE DETECTION OF GRAPEVINE FANLEAF VIRUS

采用蛋白A抗体夹心和异种动物抗体夹心两种间接酶联免疫吸附试验(ELISA)直接检测葡萄叶片和试管苗中的葡萄扇叶病毒,使试验程序简化,灵敏度与双抗体夹心ELISA直接法相当或较高。检测结果表明,经检测的103样品,其中田间品种扇叶病毒带毒率高达74.3%,热处理试管苗为5.6%,国外引进的脱毒品种(系)也有17.7%带毒。

Grapevine fanleaf virus (GFV) Gx and DIL isolates were purified from infected Chenopodium quinoa leaves. The production was 1.8mg virus per 100g leaf tissue. Two forms of indirect enzyme-linked immunosorbent assay (ELISA) were devised to detect GFV in leaf tissues of grapevines. Their procedures were simplified by using commercial conjugates and unfractionated antisera or ascites fluids. The sensitivity of heterogeneous animal antibody sandwich (HAS) ELISA was 1.6-8.0ng/ml purified virus, and that of protein A Sandwich (PAS) ELISA was 40ng/ml.The results showed, the GFV concentration in apical young leaves was higher than other leaves on the same shoot. Plants of 35 cultivars in the field, 34 accesions of imported vines in glasshouse, 18 plantlets from heat-treated mother plants and 16 shoot tip cultures in vitro were detected using indirect ELISA. and the percentages of GFV-infected plants were 74.3%, 17.7%, 5.6% and 50%, respectively. The effect of non-specific reaction of indirect ELISA on the detection was also studied in the experiments.